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Characterization of Acaricide Resistance, Plant-Mediated Rnai Against Two-Spotted Spider Mites (Tetranychus Urticae Koch), and Assessing Off- and Non-Target Effects

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Characterization of Acaricide Resistance, Plant-Mediated Rnai Against Two-Spotted Spider Mites (Tetranychus Urticae Koch), and Assessing Off- and Non-Target Effects Western University Scholarship@Western Electronic Thesis and Dissertation Repository 10-4-2017 11:30 AM Characterization of Acaricide Resistance, Plant-Mediated RNAi Against Two-Spotted Spider Mites (Tetranychus urticae Koch), and Assessing Off- and Non-Target Effects Hooman Hosseinzadeh Namin The University of Western Ontario Supervisor Dr. Vojislava Grbic The University of Western Ontario Joint Supervisor Dr. Ian Scott The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy ©Follow Hooman this and Hosseinzadeh additional works Namin at: https:/ 2017/ir .lib.uwo.ca/etd Part of the Agricultural Science Commons, Biology Commons, Cell Biology Commons, Genetics Commons, Molecular Genetics Commons, Plant Biology Commons, and the Plant Breeding and Genetics Commons Recommended Citation Hosseinzadeh Namin, Hooman, "Characterization of Acaricide Resistance, Plant-Mediated RNAi Against Two-Spotted Spider Mites (Tetranychus urticae Koch), and Assessing Off- and Non-Target Effects" (2017). Electronic Thesis and Dissertation Repository. 4922. https://ir.lib.uwo.ca/etd/4922 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The two-spotted spider mite (TSSM), Tetranychus urticae (Koch), is one of the most damaging agricultural pests in the world. It feeds on over 150 crops, causing considerable yield losses in greenhouses and agricultural fields. Currently, using synthetic acaricides is the main method to control TSSM. However, it can develop resistance to acaricides with repeated exposure, and typically resistance can occur within two to four years. To understand the underlying mechanisms of spider mite adaptation to acaricides is an essential part of resistance management strategy. The resistance ratio of the pyridaben-selected strain compared with the pre-selection strain was estimated at greater than 3000-fold. The results of synergism experiments indicated that detoxification by cytochrome P450 enzymes may be the major resistance mechanism to pyridaben by the spider mite population studied. Piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenases, can be used as an effective synergist to control pyridaben resistant populations of spider mites. RNA interference (RNAi) can be applied as an alternative pest management strategy with less likelihood of developing resistance. Plant-derived Vacuolar-type H+-ATPase RNAi through dsRNA ingestion in spider mites resulted in a significant decrease of the level of V-ATPase transcripts by 36%, while fecundity was reduced by 28%. Unwanted non-target effects often hamper the application of plant-derived RNAi pest management. However, no non-target RNAi effect on mortality and gain weight of cabbage looper and green peach aphid exposed to dsRNAs targeting Vacuolar-type H+-ATPase RNAi through dsRNA ingestion was observed. These results represent a potential delivery method of dsRNA to control spider mites through transgenic plants. Keywords Tetranychus urticae, Pyridaben, METI acaricides, Cytochrome P450, Arabidopsis, RNA interference, Transgenic plants, hpRNA, Vacuolar-type H+-ATPase, Off-Target effect, Non- target effect. i Co-Authorship Statement A part of Chapter 3 was published in PLoS One1, that was coauthored by Dr. Vojislava Grbić, Dr. Miodrag Grbić, Dr. Pierre Hilson, Dr. Vladimir Zhurov, Dr. Takeshi Suzuki, Dr. Maria Andreia Nunes, Dr. Maria Urizarna Espana, Dr. Tawhid Rahman, Nicolas Bensoussan, Pengyu Jin, and Rebecca De Clercq. The efficiency of RNAi knock-down in Tetranychus urticae using five methods of double-stranded RNA (dsRNA) delivery was compared. These methods include dsRNA ingestion through floating leaves on a dsRNA solution, dsRNA- expressing plants, artificial diet supplemented with dsRNA, dsRNA-coated leaves, and dsRNA soaking. Spider mite TuVATPase transgene was cloned and transformed into Arabidopsis thaliana plants by Dr. Pierre Hilson, and homozygous seed selection experiments were performed by Dr. Tawhid Rahman. I performed the rest of the plant derived RNAi experiments, analyzed data, and wrote the manuscript for the dsRNA- expressing plants delivery method, while my supervisor secured funding, wrote and polished the final manuscript, and submitted it to the journal. 1Suzuki T., Nunes M.A., España M.U., Namin H.H., Jin P., Bensoussan N., Zhurov V., Rahman T., De Clercq R., Hilson P., Grbić V. Grbić M. 2017. RNAi-based reverse genetics in the chelicerate model Tetranychus urticae: A comparative analysis of five methods for gene silencing. PLoS ONE, 12 (7), e0180654. ii Acknowledgments I am extremely grateful to my supervisors Dr. Vojislava Grbić and Dr. Ian Scott for granting me the opportunity to pursue a Ph.D. degree in their labs. I greatly appreciate their patience, encouragement, and excellent advice throughout my academic career. I would like to thank my advisory research committee, Dr. Jeremy McNeil and Dr. Brent Sinclair, for their constructive criticism and guidance throughout my project. I would also like to express my appreciation to Dr. Mike Grbić and Dr. Vladimir Zhurov for their valuable comments and guidance in the laboratory. Special thanks to Mrs. Biljana Popovic and Mr. Igor Lalin for their technical support during my research project. My special thanks to all the past and present members of Dr. Grbić’s and Dr. Scott’s lab for their friendship, support and academic feedbacks. I am deeply indebted to my remarkable parents and my brother for their wonderful support and encouragement throughout my life. This thesis is dedicated to the memory of my father, Dr. Abolfath Hosseinzadeh Namin, who was a constant source of inspiration and moral support, and taught me the value of hard work and education. This project was supported financially by the Government of Canada through the Ontario Research Fund-Research Excellence Round 8 (RE08-067) to Dr. M. Grbić and Dr. V. Grbić, and the University of Western Ontario through the Western Strategic Support program to Dr. V. Grbić. Lastly, I would like to offer my blessing to all of those who supported me for the completion of my project, as well as to express my apologies that I could not mention them personally one by one. iii Table of Contents Abstract ................................................................................................................................ i Co-Authorship Statement.................................................................................................... ii Acknowledgments.............................................................................................................. iii List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1 ............................................................................................................................. 1 1 General Introduction ...................................................................................................... 1 1.1 Tetranychus urticae biology, life cycle and host plants ......................................... 1 1.2 Crop damage of Tetranychus urticae ...................................................................... 3 1.3 Acaricide as a predominant control strategy of TSSM ........................................... 4 1.4 Mechanisms of insecticide resistance ..................................................................... 5 1.5 The requirements for integrated pest management (IPM) of TSSM ...................... 7 1.6 Introduction to RNA interference ........................................................................... 8 1.7 Methods of dsRNA delivery ................................................................................... 9 1.8 Plant-derived RNAi as a novel pest management approach ................................. 11 1.9 RNAi off-target and non-target effects ................................................................. 13 1.10 Rationale and objectives ....................................................................................... 14 1.11 References ............................................................................................................. 18 Chapter 2 ........................................................................................................................... 37 2 Molecular and metabolic mechanisms of pyridaben resistance in TSSM ................... 37 2.1 Introduction ........................................................................................................... 37 2.1.1 Mitochondrial Electron Transport Inhibitor (METI) acaricides ............... 37 2.1.2 METI acaricide classes ............................................................................. 38 2.1.3 Resistance mechanisms to METI acaricides ............................................. 39 iv 2.1.4 Objectives ................................................................................................. 41 2.2 Material and methods ............................................................................................ 42 2.2.1 Acaricides and enzyme
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